Constructs for delivery of therapeutic agents to neuronal cells

ABSTRACT

A non-toxic polypeptide, for delivery of a therapeutic agent to a neuronal cell, comprises a binding domain that binds to the neuronal cell, and a translocation domain that translocates the therapeutic agent into the neuronal cell, wherein the translocation domain is not a H N  domain of a clostridial toxin and is not a fragment or derivative of a H N  domain of a clostridial toxin.

This application is a 371 of PCT/GB00/04644, filed on Dec. 4, 2000, andpublished in English on Aug. 16, 2001.

The present invention relates to constructs for delivering therapeuticsubstances to neuronal cells, to manufacture and use thereof, and inparticular to constructs based on clostridial neurotoxins.

There are presently few effective treatments for major disorders of thecentral nervous system. Such disorders include neurodegenerativediseases, stroke, epilepsy, brain tumours, infections and HIVencephalopathy, and sufferers of these diseases far outnumber themorbidity of cancer and heart disease. The number of sufferers for CNSdisorders such as stroke and the neurodegenerative diseases is set togrow, particularly in developed countries where the average age of thepopulation is increasing. As our understanding of brain pharmacologyincreases and the underlying pathologies of diseases are elucidated,potential therapeutic strategies become apparent. All these treatments,however, face the formidable problem of efficient delivery oftherapeutics to the various neuronal cell populations involved. Vectorswhich can effect efficient delivery to neuronal cells are thus requiredfor a broad range of therapeutic substances, including drugs, enzymes,growth factors, therapeutic peptides and genes.

Ischemia/reperfusion injury induced by stroke or injury is one notableexample in which rapid and efficient delivery of therapeutic agentswould afford considerable benefit. Neurons injured by trauma or ischemiaproduce elevated levels of free oxygen radicals and release large amountof glutamate. These substances in high concentration are toxic to bothneurons and surrounding cells which potentiate and amplify the damageprocess. Agents such as superoxide dismutase or glutamine synthetasewhich reduce the levels of these toxic substances have been shown toreduce neuronal cell death in a variety of in vitro and in vivo ischemiamodels (Gorovits et al. PNAS (1997) 94, 7024-7029; Francis et al.Experimental Neurology (1997) 146, 435-443; Lim et al. Ann. Thorac.Surg. (1986) 42, 282-286; Cuevas et al. Acta Anat. (1990) 137, 303-310).A major problem in the use of such therapies is the delivery of usefulconcentrations of the active agent to the site of trauma. Specificneuronal vectors could therefore play an important role in targetingsuch compounds to neuronal cells.

Peripheral nervous system disorders, such as motor neuron disease, arefurther examples of diseases which would benefit from the targeteddelivery of therapeutic agents. Such therapies could take the form ofdrug delivery or DNA delivery via gene therapy strategies.

Gene therapy holds considerable promise for the treatment ofneurodegenerative diseases such as Parkinson's and Alzheimer's diseases.Most of the currently available viral and non-viral gene deliveryvectors lack tissue specificity which reduces both their efficiency andsafety of use. Suitable neuronal cell-specific targeting ligands aretherefore required for a broad range of gene vectors to enable effectivetreatments for neuronal diseases to be developed.

The botulinum neurotoxins are a family of protein toxins whose primarysite of action is the neuromuscular junction where they block therelease of the transmitter acetylcholine. The action of these toxins onthe peripheral nervous system of man and animals results in the syndromebotulism, which is characterised by widespread flaccid muscularparalysis (Shone (1986) in ‘Natural Toxicants in Foods’, Editor D.Watson, Ellis Harwood, UK). Each of the botulinum neurotoxins consistsof two disulphide-linked subunits; a 100 kDa heavy subunit which plays arole in the initial binding and internalisation of the neurotoxin intothe nerve ending (Dolly et. al. (1984) Nature, 307, 457-460) and a 50kDa light subunit which acts intracellularly to block the exocytosisprocess (McInnes and Dolly (1990) Febs Lett., 261, 323-326; de Paiva andDolly (1990) Febs Lett., 277, 171-174).

The clostridial neurotoxins are potent inhibitors of calcium-dependentneurotransmitter secretion in neuronal cells. They are currentlyconsidered to mediate this activity through a specific endoproteolyticcleavage of at least one of three vesicle or pre-synaptic membraneassociated proteins VAMP, syntaxin or SNAP-25 which are central to thevesicle docking and membrane fusion events of neurotransmittersecretion. The neuronal cell targeting of tetanus and botulinumneurotoxins is considered to be a receptor mediated event followingwhich the toxins become internalised and subsequently traffic to theappropriate intracellular compartment where they effect theirendopeptidase activity.

Clostridial neurotoxins share a common architecture of a catalyticL-chain (LC, ca 50 kDa) disulphide linked to a receptor binding andtranslocating H-chain (HC, ca 100 kDa). The HC polypeptide is consideredto comprise all or part of two distinct functional domains. Thecarboxy-terminal half of the HC, termed the H_(C) domain (ca 50 kDa), isinvolved in the high affinity, neurospecific binding of the neurotoxinto cell surface receptors on the target neuron, whilst theamino-terminal half, termed the H_(N) domain (ca 50 kDa), is consideredto mediate the translocation of at least some portion of the neurotoxinacross cellular membranes such that the functional activity of the LC isexpressed within the target cell. The H_(N) domain also has theproperty, under conditions of low pH, of forming ion-permeable channelsin lipid membranes, and this may in some manner relate to itstranslocation function. For botulinum neurotoxin type A (BoNT/A) thesedomains are considered to reside within amino acid residues 872-1296 forthe H_(C), amino acid residues 449-871 for the H_(N) and residues 1-448for the LC.

It is therefore possible to provide functional definitions of thedomains within the neurotoxin molecule, as follows:—

-   -   (A) clostridial neurotoxin light chain:—        -   a metalloprotease exhibiting high substrate specificity for            vesicle and/or plasma membrane associated proteins involved            in the exocytotic process. In particular, it cleaves one or            more of SNAP-25, VAMP (synaptobrevin/cellubrevin) and            syntaxin.    -   (B) clostridial neurotoxin heavy chain H_(N) domain:—        -   a portion of the heavy chain which enables translocation of            that portion of the neurotoxin molecule such that a            functional expression of light chain activity occurs within            a target cell.        -   the domain responsible for translocation of the            endopeptidase activity, following binding of neurotoxin to            its specific cell surface receptor via the binding domain,            into the target cell.        -   the domain responsible for formation of ion-permeable pores            in lipid membranes under conditions of low pH.    -   (c) clostridial neurotoxin heavy chain H_(C) domain:        -   a portion of the heavy chain which is responsible for            binding of the native holotoxin to cell surface receptor(s)            involved in the intoxicating action of clostridial toxin            prior to internalisation of the toxin into the cell.

The identity of the cellular recognition markers for these toxins iscurrently not understood and no specific receptor species have yet beenidentified although Kozaki et al. have reported that synaptotagmin maybe the receptor for botulinum neurotoxin type B. It is probable thateach of the neurotoxins has a different receptor.

Tetanus toxin is structurally very similar to botulinum neurotoxins butits primary site of action is the central nervous system where it blocksthe release of inhibitory neurotransmitters from central synapses(Renshaw cells).

Tetanus and the botulinum neurotoxins from most of the seven serotypes,together with their derived heavy chains, have been shown to bind a widevariety of neuronal cell types with high affinities in the nM range,e.g. botulinum type B neurotoxin (Evans et al. (1986) Eur. J. Biochem.154, 409-416).

However, a major obstacle to the use of the native clostridial heavychain fragments as delivery vectors is that their highly aggregatedstate in solution prevent their adequate diffusion into body tissue andhence reduces their efficiency as targeting vectors. A furthersignificant problem with any proposed clinical use of native tetanustoxin fragments as neuronal targeting ligands for therapeutics is theexistence of circulating antibodies to the toxin in the majority of thepopulation who have been immunized against tetanus. The presence ofthese antibodies is likely to reduce the efficacy of constructs based ontetanus toxin fragments. Thus, clostridial neurotoxin fragments do notoffer solutions to the problems identified.

The present invention is based upon the discovery of the practicaldifficulties in using clostridial neurotoxin-based therapeuticcompositions, and the devising of modified polypeptides and hybridpolypeptides based on clostridial neurotoxin fragments that avoid theaforementioned drawbacks.

Accordingly, a first aspect of the invention provides a non-toxicpolypeptide, for delivery of a therapeutic agent to a neuronal cell,comprising:—

-   -   a binding domain that binds to the neuronal cell, and    -   a translocation domain that translocates the therapeutic agent        into the neuronal cell,        wherein the translocation domain is not a H_(N) domain of a        clostridial neurotoxin and is not a fragment or derivative of a        H_(N) domain of a clostridial toxin.

The binding domain is suitably comprised of or derived from clostridialheavy chain fragments or modified clostridial heavy chain fragments. Asused herein, the term “modified clostridial heavy chain fragment” meansa polypeptide fragment which retains similar biological functions to thecorresponding heavy chain of a botulinum or tetanus neurotoxin butdiffers in its amino acid sequence and other properties compared to thecorresponding heavy chain. The invention more specifically provides suchconstructs which are based on fragments derived from botulinum andtetanus neurotoxins.

In a further aspect, the invention also provides a polypeptide, fordelivery of a therapeutic agent to a neuronal cell, comprising:—

-   -   a binding domain that binds to the neuronal cell, and    -   a translocation domain that translocates the therapeutic agent        into the neuronal cell,        wherein the resulting polypeptide construct is non-aggregating.

Whether the construct is an aggregating one is usually apparent from alack of solubility of the construct, and this may be seen upon simplevisual inspection of the construct in aqueous media: non-aggregatingdomains result in constructs of the invention that are partially orpreferably totally soluble whereas aggregating domains result innon-soluble aggregates of polypeptides having apparent sizes of manytens or even hundreds the size of a single polypeptide. Generally, theconstruct should be non-aggregating as measured by size on gelelectrophoresis, and the size or apparent size of the construct measuredshould preferably be less than 5.0×10⁵ daltons, more preferably lessthan 1.5×10⁵ daltons, with the measuring being suitably carried out onnative PAGE using physiological conditions.

A still further aspect of the invention provides a polypeptide, fordelivery of a therapeutic agent to a neuronal cell, comprising:—

-   -   a binding domain that binds to the neuronal cell, and    -   a translocation domain that translocates the therapeutic agent        into the neuronal cell,        wherein the translocation domain is selected from (1) a H_(N)        domain of a diphtheria toxin, (2) a fragment or derivative        of (1) that substantially retains the translocating activity of        the H_(N) domain of a diphtheria toxin, (3) a fusogenic        peptide, (4) a membrane disrupting peptide, (5) a H_(N) from        botulinum toxin C₂ and (6) translocating fragments and        derivatives of (3), (4) and (5).

It is to be noted that botulinum toxin C₂ is not a neurotoxin as it hasno neuronal specificity, instead it is an enterotoxin and suitable foruse in the invention to provide a non-aggregating translocation domain.

A yet further aspect of the invention provides a polypeptide, fordelivery of a therapeutic agent to a neuronal cell, comprising:—

-   -   a binding domain that binds to the neuronal cell, and    -   a translocation domain that translocates the therapeutic agent        into the neuronal cell,        wherein the polypeptide has reduced affinity to neutralising        antibodies to tetanus toxin compared with the affinity to such        antibodies of native tetanus toxin heavy chain.

The above aspects may singly or in any combination be exhibited bypolypeptides of the invention and thus a typical preferred polypeptideof the invention (i) lacks the neurotoxic activities of botulinum andtetanus toxins, (ii) displays high affinity to neuronal cellscorresponding to the affinity of a clostridial neurotoxin for thosecells, (iii) contains a domain which can effect translocation acrosscell membranes, and (iv) occurs in a less aggregated state than thecorresponding heavy chain from botulinum or tetanus toxin inphysiological buffers.

A significant advantage of the polypeptides of the invention is theirnon-aggregated state, thus rendering them usable as soluble polypeptideswhere the prior art constructs were not and overcoming most if not allof the drawbacks of previous constructs based upon clostridialneurotoxins.

The polypeptides according to the invention generally include sequencesfrom the H_(C) domains of the botulinum and tetanus neurotoxins andthese are combined with functional domains from other proteins, suchthat the essential functions of the native heavy chain, binding toneuronal cells, is retained. Thus, for example, the H_(C) domain ofbotulinum type F neurotoxin is fused to the translocation domain derivedfrom diphtheria toxin to give a modified clostridial heavy chainfragment. Surprisingly, such polypeptides are more useful as constructsfor delivering substances to neuronal cells than are the nativeclostridial heavy chains.

Thus, according to a preferred aspect of the invention there is provideda polypeptide having an amino acid sequence comprising (a) asub-sequence based on the H_(C) fragment of botulinum or tetanusneurotoxin, and (b) a sub-sequence based on a translocation domain, e.g.from diphtheria toxin, that is not derived from a clostridialneurotoxin, and wherein the said polypeptide (i) lacks the neurotoxinactivities of botulinum and tetanus toxins, (ii) displays high affinityto neuronal cells, (iii) contains a domain which can effecttranslocation across cell membranes and (iv) occurs in a less aggregatedstate than the corresponding heavy chain of botulinum or tetanus toxinin physiological buffers.

The modified clostridial heavy chain is suitably produced by combiningthe binding domain (H_(C) domain) of a clostridial neurotoxin with anon-clostridial translocation domain. Thus, for example, a modifiedclostridial heavy chain fragment may be constructed from thetranslocation domain of diphtheria toxin (residues 194-386) fused to theH_(C) domain of a botulinum toxin (e.g. type F H_(C) fragment, residues865-1278; type A H_(C) fragment, residues 872-1296).

In another embodiment of the invention, the modified clostridial heavychain is produced by combining the H_(C) domain of a clostridialneurotoxin with a membrane disrupting peptide which functions as atranslocation domain, suitably a viral peptide. Thus, for example, amodified clostridial heavy chain fragment may be constructed bycombining the H_(C) domain of a botulinum toxin with a peptide based oninfluenza virus haemagglutinin HA2 (residues 1-23).

The polypeptides of the invention have properties which make them usefulas neuronal targeting ligands; they are non-toxic and yet retain thespecific, high affinity binding to neuronal cells displayed by thebotulinum or tetanus toxins. Unlike the native clostridial heavy chains,however, the modified clostridial heavy chains occur in a lessaggregated state in solution which improves their access to neuronalcells. The preferred constructs are soluble in aqueous solution, incontrast to the highly aggregated state of the prior art constructs.

In another aspect of the invention, there is provided a modified tetanusheavy chain fragment which, in addition to the properties of modifiedheavy chains defined above, has the added advantage in that it hasreduced affinity to neutralizing antibodies, present as a result ofanti-tetanus inoculation, compared to the native tetanus toxin heavychain. The polypeptides according to this aspect of the inventiongenerally include subsequences derived from the heavy chain of tetanustoxin (residues 458-1315) and from which epitopes responsible for theimmunogenicity of tetanus toxin have optionally been reduced or removed.Thus, for example, it is desirable to eliminate immunogenic epitopesassociated with H_(C) domain as well as that of the H_(N) domain.Although it is possible to eliminate epitopes by deleting small numbersof amino acids (e.g. less than 20 or preferably less than 10 aminoacids), it has been found that epitopes associated with immunogenicityof tetanus toxin heavy chain can be reduced more rigorously by replacinga large number of amino acid residues (e.g. at least 100, at least 200and preferably 400 or more residues) with amino acid sequences fromother toxins.

Thus according to a preferred aspect of the invention related tomodified tetanus heavy chains, there is provided a polypeptide having anamino sequence comprising (a) an H_(N) domain derived from anon-clostridial source (e.g. diphtheria toxin), (b) one or moresubsequences derived from the sequence of a botulinum H_(C), and (c) oneor more subsequences derived from the sequence of tetanus toxin H_(C),and wherein said polypeptide (i) lacks the neurotoxin activities ofbotulinum and tetanus toxins, (ii) displays high affinity to neuronalcells corresponding to the neuronal binding of tetanus neurotoxin, (iii)contains a domain which can effect translocation across cell membranesand (iv) has low affinity to neutralizing antibodies to tetanus toxinwhich are present as result of anti-tetanus inoculation.

This latter modified tetanus heavy chain fragment can be produced bycombining the binding domain (H_(C) domain) of tetanus neurotoxin with anon-clostridial translocation domain. Thus, for example, a modifiedtetanus heavy chain fragment may be constructed from the translocationdomain of diphtheria toxin (residues 194-386) fused to the H_(C) domainof a tetanus toxin (residues 865-1315).

In another embodiment of the invention the modified tetanus heavy chainis derived a non-clostridial translocation domain fused to the H_(C)domain of a botulinum toxin into which the minimal domains of tetanustoxin are inserted to confer tetanus toxin-like binding activity ontothe resulting hybrid. Thus, for example a modified tetanus heavy chainmay be constructed from the translocation domain of diphtheria toxin(residues 194-386) fused to the H_(C) domain of a botulinum type Ffragment (residues 865-1278) in which residues 1097-1273 of the latterhave been replaced by homologous sequences from tetanus toxin.

The modified tetanus heavy chains have properties which make them usefulas neuronal targeting ligands; they are non-toxic and yet retain thespecific, high affinity binding to neuronal cells displayed by tetanustoxin. Unlike native tetanus toxin binding fragments, however, themodified clostridial binding fragments have different immunogenicproperties which makes them more useful clinically. Specifically, thedifferent immunogenic properties of the modified clostridial bindingfragments of the invention significantly reduce the problems caused byexisting antibodies to native tetanus toxin sequences.

While the use of modified heavy chains based on botulinum neurotoxins asneuronal targeting ligands does not suffer from the problem ofpre-existing circulating antibodies, tetanus toxin is unique amongst theclostridial toxins in that it has selectivity to inhibitory neurons(e.g. Renshaw cells) and as such the modified tetanus toxin heavy chainsare valuable targeting ligands for this class of neuron. Tetanus toxinalso has the property that it can retrograde transport from theperipheral to the central nervous system.

In another embodiment of the invention, the modified clostridial heavychain fragment is fused to a linker peptide via the N-terminus of thetranslocation domain to which a polypeptide payload may be attached. Anexamples of such a linker peptide is the sequence CGLVPAGSGP (SEQ IDNO:1) which contains the thrombin protease cleavage site and a cysteineresidue for disulphide bridge formation. Such a peptide linker allowsproduction of a recombinant fusion protein comprising a polypeptidetherapeutic molecule fused by the linker peptide to the N-terminus ofthe modified clostridial heavy chain fragment. The latter single chainfusion protein may then be treated with thrombin to give a dichainprotein in which the polypeptide therapeutic is linked to thetranslocation domain of the modified clostridial heavy chain fragment bya disulphide link. In another example of a linker peptide in which thetranslocation domain does not contain a free cysteine residue near itsC-terminus, such as is the case when the translocation domain is afusogenic peptide, the linker peptide contains both cysteine residuesrequired for the disulphide bridge. An example of the latter linkerpeptide is the amino acid sequence: CGLVPAGSGPSAGSSAC (SEQ ID NO:2).

In another embodiment of the invention, the modified clostridial heavychain is linked to a polypeptide which may be an enzyme, growth factor,protein or peptide which has therapeutic benefits when delivered toneuronal cells. The polypeptide may be linked to the modifiedclostridial heavy chain by chemical means. Alternatively the polypeptidemay be produced as a fusion protein linked to the modified clostridialbinding fragment by recombinant technology using the linker peptides asdescribed above. In such an example, the construct would contain thefollowing components:—

-   -   a polypeptide therapeutic substance;    -   a linker peptide; and    -   a modified clostridial heavy chain

An example of a polypeptide therapeutic payload is superoxide dismutase.

In yet another embodiment of the invention, the modified clostridialheavy chain is linked directly or indirectly to DNA such that theconstruct is capable of delivering the DNA to neuronal cells, e.g. viathe receptor for tetanus toxin. Such constructs have gene therapyapplications and be used to switch on, or off, selected genes with thecell. The DNA may be contained within a liposome or be condensed via apeptide or protein. The modified clostridial heavy chain may bechemically linked to the protein that effects the DNA condensation bychemical coupling agents. Alternatively, the modified clostridial heavychain may be produced as a fusion protein, by recombinant technology,with a peptide that can effect the condensation of DNA.

In yet another embodiment of the invention, the modified clostridialheavy chain fragment may be linked to a recombinant virus such that themodified virus has an altered tropism and is capable of transducingcells via the tetanus toxin receptor. Such a construct is of use tocorrect genetic defects within neuronal cells by switching on, or off,selected genes. The modified clostridial heavy chain fragment may belinked directly to the surface of the virus using chemical cross-linkingagents. Alternatively the modified clostridial heavy chain fragment maybe linked to the recombinant virus via an antibody which specificallybind to the virus. In this instance the modified clostridial bindingfragment is chemically coupled to a polyclonal or monoclonal antibodywhich specifically recognizes a marker on the surface of the virus. Asimilar modified clostridial binding fragment-antibody fusion proteincould be produced by recombinant technology in which the antibodycomponent is a recombinant single chain antibody.

In yet another embodiment of the invention, the modified clostridialheavy chain fragment is linked to a drug release system such as amicroparticle constructed from a suitable polymer, e.g. poly(lactide-co-glycolide), polyhydroxylalkonate, collagen,poly(divinyl-ether-comaleic anhydride, poly (styrene-co-maleicanhydride) or other polymer useful in such microparticles. The modifiedclostridial heavy chain fragment may be linked to the drug releasesystem by covalent chemical coupling, or electrostatic or hydrophobicforces. The modified clostridial heavy chain fragment may also beencapsulated within the release vehicle together with the therapeuticpayload provided that a portion of the modified clostridial bindingfragment is exposed at the surface. Alternatively, the modifiedclostridial heavy chain fragment may be linked, at either the N- orC-terminal end, to a peptide or protein to facilitate coupling of thefragment to the drug release system.

Other strategies are known by which modified heavy chain bindingfragments can be linked to range of therapeutic substances using avariety of established chemical cross-linking techniques, and a varietyof fusion proteins can be produced containing a modified clostridialbinding fragment and another polypeptide. Using these techniques avariety of substances can be targeted to neuronal cells using themodified clostridial binding fragments. Examples of possible uses of themodified clostridial binding fragments as neuronal delivery vectors aregiven in more detail below in Table 1.

Constructs of the invention may be introduced into either neuronal ornon-neuronal tissue using methods known in the art. By subsequentspecific binding to neuronal cell tissue, the targeted construct exertsits therapeutic effects. Ideally, the construct is injected near a siterequiring therapeutic intervention.

The construct of the invention may be produced as a suspension,emulsion, solution or as a freeze dried powder depending on theapplication and properties of the therapeutic substance. The constructof the invention may be resuspended or diluted in a variety ofpharmaceutically acceptable liquids depending on the application.

“Clostridial neurotoxin” means either tetanus neurotoxin or one of theseven botulinum neurotoxins, the latter being designated as serotypes A,B C₁, D, E, F or G.

“Modified clostridial heavy chain fragment” means a polypeptide fragmentwhich binds to neuronal cell receptors in similar manner to acorresponding heavy chain derived from botulinum or tetanus toxins butdiffers in its amino acid sequence and properties compared to thecorresponding fragment derived from tetanus toxin.

“Bind” in relation to the botulinum and tetanus heavy chain fragments,means the specific interaction between the clostridial fragment and oneor more cell surface receptors or markers which results in localizationof the binding fragment on the cell surface. In the case of theclostridial neurotoxins, the property of a fragment being able to ‘bind’like a fragment of a given serotype can be demonstrated by competitionbetween the ligand and the native toxin for its neuronal cell receptor.

“High affinity binding specific to neuronal cell corresponding to thatof a clostridial neurotoxin” refers to the ability of a ligand to bindstrongly to cell surface receptors of neuronal cells that are involvedin specific binding of a given neurotoxin. The capacity of a givenligand to bind strongly to these cell surface receptors may be assessedusing conventional competitive binding assays. In such assaysradiolabelled clostridial neurotoxin is contacted with neuronal cells inthe presence of various concentrations of non-radiolabelled ligands. Theligand mixture is incubated with the cells, at low temperature (0-3° C.)to prevent ligand internalization, during which competition between theradiolabelled clostridial neurotoxin and non-labelled ligand may occur.In such assays when the unlabelled ligand used is the same as that ofthe labelled neurotoxin, the radiolabelled clostridial neurotoxin willbe displaced from the neuronal cell receptors as the concentration ofnon-labelled neurotoxin is increased. The competition curve obtained inthis case will therefore be representative of the behaviour of a ligandwhich shows “high affinity binding specificity to neuronal cellscorresponding to that of a clostridial neurotoxin”, as used herein.

“Translocation domain” means a domain or fragment of a protein whicheffects transport of itself and/or other proteins and substances acrossa membrane or lipid bilayer. The latter membrane may be that of anendosome where translocation will occur during the process ofreceptor-mediated endocytosis. Translocation domains can frequently beidentified by the property of being able to form measurable pores inlipid membranes at low pH (Shone et al. Eur J. Biochem. 167, 175-180).Examples of translocation domains are set out in more detail below inFIG. 1. In the application, translocation domains are frequentlyreferred to as “H_(N) domains”.

“Translocation” in relation to translocation domain, means theinternalization events which occur after binding to the cell surface.These events lead to the transport of substances into the cytosol ofneuronal cells.

“Therapeutic substances” or “agents” mean any substance, agent ormixture thereof, which, if delivered by the modified clostridial bindingfragment, would be beneficial to the treatment of neuronal diseases.Examples of these include drugs, growth factors, enzymes, and DNApackaged in various forms (e.g. modified viruses, cationic liposomes,and condensed DNA).

Also provided in the present invention are methods of manufacture of thepolypeptides of the invention by expressing in a host cell a nucleicacid encoding the polypeptide, and the use of a polypeptide or acomposition according to the invention in the treatment of a diseasestate associated with neuronal cells.

The invention is now illustrated in the following specific embodimentsand accompanied by drawings in which:—

FIG. 1 shows modified clostridial heavy chain fragments produced byrecombinant technology as a fusion proteins;

FIG. 2 shows modified clostridial heavy chain fragments produced byrecombinant technology; fusion proteins may contain one or morepurification peptide tags to assist in the purification of the protein;one or more protease cleavage sites may also be included to enableremoval of the purification peptide tags; similar purificationstrategies may also be employed for modified clostridial bindingfragments containing a translocation domain;

FIG. 3 shows linkage of a modified clostridial binding fragment to atherapeutic substance; the modified clostridial heavy chain contains atranslocation domain which has a free thiol group (an example oftranslocation domain with this property is amino acid sequence 194-386of diphtheria toxin), a free amino group on the therapeutic substance ismodified with a cross-linking reagent (e.g. SPDP; Pierce & Warriner, UKLtd.) which will subsequently allow conjugate formation using the freethiol present on the modified clostridial binding fragment;

FIG. 4 shows the formation of a conjugate between a modified clostridialheavy chain fragment and an oligonucleotide as described in Example 4;

FIG. 5 shows a strategy for producing a recombinant modified clostridialheavy chain as a fusion protein with a polypeptide therapeuticsubstance. The latter is fused to the modified clostridial heavy chainby a linker peptide. The linker peptide contains a unique proteasecleavage site (e.g. that recognized by thrombin) and a cysteine residue.Examples of linker peptides are (a) CGLVPAGSGP; and (b) CGIEGRAPGP (SEQID NO:18). The cysteine residue forms a disulphide bridge with ananother available cysteine residue on the translocation domain of themodified heavy chain fragment. If desirable, then by treatment withthrombin, a dichain product may be produced in which the polypeptidetherapeutic substance is linked to the heavy chain via a disulphidebridge;

FIG. 6 shows a comparison of the binding of a modified heavy chain withthat of the native neurotoxin to neuronal synaptic membranes, themodified heavy chain displaying the binding characteristics of tetanusneurotoxin as assessed by the method described in Example 7;

FIG. 7 shows the binding to neuronal membranes of a modified clostridialheavy chain based on the binding domain of botulinum type F neurotoxin;in this example, modified heavy chain contained the translocation(H_(N)) domain of diphtheria toxin and the binding (H_(c)) domain oftype F neurotoxin; and

FIG. 8 shows a comparison of the molecular sizes, under non-denaturingconditions, of a modified clostridial heavy chain compared to a nativeheavy chain; the modified clostridial heavy chain (DiphtheriaH_(N)-BoNT/F H_(C)) runs as a monomer of approximately 70 kDa while anative heavy chain (from BoNT/A) runs as an aggregate of >500 kDa.

In more detail, FIG. 1 shows examples of embodiments of the inventionincorporating modified clostridial heavy chain fragments.

The binding domain is derived from sequences of the clostridialneurotoxins:—

-   -   (a) H_(C) domains, e.g.        -   BoNT/A residues 872-1296        -   BoNT/B residues 859-1291        -   BoNT/C residues 867-1291        -   BoNT/D residues 863-1276        -   BoNT/E residues 846-1252        -   BoNT/F residues 865-1278        -   BoNT/G residues 864-1297        -   Tetanus residues 880-1315    -   (b) Hybrid H_(C) domains, e.g.        -   hybrids of the H_(C) domain of BoNT/F and tetanus    -   (c) Truncated H_(C) domains

The translocation domain may be derived from a number of sources:—

-   -   (a) Bacterial toxins, e.g. diphtheria toxin fragment B (residues        194-386)    -   (b) Viral fusogenic peptides, e.g. from influenza virus        haemagglutinin HA-2    -   (c) Synthetic membrane disrupting peptides (e.g. Plank et        al., J. Biol. Chem., 269, 12918-12924).

FIG. 2 shows examples of Recombinant Modified Clostridial Heavy ChainFragment Fusion Proteins Showing Positions of Purification Peptide Tagsand Specific Protease Cleavage Sites (by treatment with the appropriateprotease, the purification peptide tags may be removed from the modifiedclostridial binding fragment).

Examples of purification peptides tags are:

-   -   His6    -   S peptide    -   T7 peptide    -   Calmodulin binding peptide    -   Maltose binding protein

Examples of specific protease cleavage sites are:—

-   -   Thrombin    -   Enterokinase    -   Factor X

EXAMPLE 1

Preparation and Purification of a Recombinant Modified Clostridial HeavyChain Fragments.

Standard molecular biology protocols were used for all geneticmanipulations (e.g. Sambrook et al. 1989, Molecular Cloning a LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). An entirely synthetic gene encoding the H_(C) regions ofbotulinum toxin from C. botulinum type F (residues 865-1278) and tetanustoxin (residues 880-1315) were generated using Recursive PCR reactions(Prodromou & Pearl 1992, Protein Engineering, 5: 827-829) usingself-priming oligonucleotides containing the desired sequence. The codonbias and GC/AT base ratio was adjusted for ease of expression in E.coli. Fragments were cloned sequentially into pLitmus 38 (New EnglandBiolabs, Inc., Beverly, Mass.) to assemble the entire gene. Constructsfor expression were sub-cloned into pMALc2 (NEB) replacing theBamH1-EcoR1 fragment. The ligation reactions were transformed into E.coli JM109 (Promega).

Plasmid DNA was amplified, purified and screened for the presence of theappropriate sequence (Ausubel et al. 1989, Current Protocols inMolecular Biology, John Wiley & Sons, New York). Gene constructionsconfirmed as possessing the correct sequences were then transformed intothe expression host E. coli BL21 (DE3) (Studier & Moffatt 1986, Journalof Molecular Biology, 189: 113-130).

Additional sequences for adding affinity purification tags and one ormore specific protease site for the subsequent removal of these affinitytags were also included in the reading frame of the gene products.

The recombinant proteins expressed in pMAL were produced withamino-terminal maltose-binding protein tags allowing proteins to bepurified by affinity chromatography on amylose resin. Briefly, culturesof E. coli BL21 (DE3) pMALc2-H_(C) were grown in Terrificbroth-ampicillin (100 μgml⁻¹)-kanamycin (30 μgml⁻¹) to an OD₆₀₀ nm of2.5-3.8, and protein expression was induced by the addition of 1 mM IPTGfor approximately 2 h. Cells were lysed by freeze/thaw followed bysonication, lysates cleared by centrifugation and supernatants loadedonto an amylose resin column and eluted with maltose. All buffers usedwere as specified by the manufacturer. Thrombin or factor Xa proteasesites were included within the protein for subsequent removal of thesepurification tags.

Other coding sequences which enable expression of the desired proteinwould also be acceptable. Other tags or linking sites may also beincorporated into the sequence. Examples of some of these options aresummarized in FIG. 2.

EXAMPLE 2

Production of a Modified Clostridial Heavy Chain Fragments.

Using the techniques described in Example 1, modified clostridial heavychain fragments was constructed by fusing domains of the H_(c) fragmentsof either botulinum type F or tetanus neurotoxins with the translocationdomain of diphtheria toxin. The amino acid sequences of examples areshown in SEQ ID NO:s 8-17, which also gives examples of modified tetanusheavy chains in which the H_(c) fragment is a hybrid of tetanus andbotulinum type F neurotoxin.

EXAMPLE 3

Coupling of a Modified Clostridial Heavy Chain Fragment to a Protein oran Enzyme.

The polypeptide, protein or enzyme to be linked to the modifiedclostridial heavy chain fragment is first derivatized with a suitablecross-linking agent. Mn-Superoxide dismutase (SOD) was modified bytreatment with a 15 molar excess of SPDP (Pierce) in 0.05M Hepes bufferpH 7.0 containing 0.15M NaCl for 60 min at 25° C. The excess SPDP wasremoved by dialysis against the same buffer At 4° C. for 16 h. Thesubstituted SOD was then mixed in a 1:5 molar ration with modifiedclostridial heavy chain fragment fused to a translocation domain derivedfrom diphtheria toxin (see FIG. 3) and incubated at 25° C. for 16 h.After incubation the SOD-modified clostridial binding fragment conjugatewas purified by gel filtration chromatography on Sephadex G200.

EXAMPLE 4

Coupling of Modified Clostridial Heavy Chain Fragment to Condensed DNA.

Poly-L-lysine (M_(r) 1000-4000) (10 mg) to be used for the condensationof DNA was dissolved in 2 ml of 20 mM Hepes buffer pH 7.4 containing0.15M NaCl (HBS). To this solution 0.6 mg of Sulpho-LC-SPDP (Pierce andWarriner, UK Ltd.) was added and the mixture incubated for 30 min at 25°C. The activated poly-L-lysine was then dialysed against HBS at 4° C.using a dialysis tubing of 1000 molecular weight cut-off and thendiluted to 1 mg/ml using HBS.

Condensation of DNA was carried out in glass tubes. Purified plasmid DNAcontaining a gene encoding a therapeutic protein (or a reporter gene)under the control of a suitable promoter (e.g. CMV immediate early, or aneuronal-specific promoter e.g. neuron-specific enolase promoter) wasmade 1 mg/ml in HBS and added to glass tubes followed by the activatedpoly-L-lysine as prepared above. Activated poly-L-lysine is added invarious proportions to the DNA (see Table 2) and incubated for 90 min at25° C.

TABLE 2 Condensation of DNA with activated poly-L-lysine. Sample no. DNA(μg) Activated Poly-L-lysine HBS 1 750 250 1500 2 1500 500 500 3 500 2501750 4 1000 500 1000

After incubation the size of the condensed DNA particles was assessedusing a Brookhaven BI90 particle sizer. The incubation conditions givingthe highest proportion of condensed DNA particle of less than 100 nM indiameter was used to produce DNA-modified clostridial binding fragmentconjugates. Modified clostridial heavy chain was dialysed against HBS.

The dialysed fragments (100 μg) was then added to 1 ml of condensed DNAand incubated for 18 h at 25° C. to from the modified clostridialbinding protein-condensed DNA construct (see FIG. 4).

EXAMPLE 5

Delivery of DNA to a Neuronal Cells via the Modified Clostridial HeavyChain Fragment Receptor.

Modified clostridial heavy chain-condensed DNA construct described inExample 4 was diluted with 2 ml MEM serum free medium. Growth media fromNG108 grown in 12 well dished was removed and 1 ml of the dilutedconstruct added and incubated for 2 h at 37° C. in the presence of 5%CO₂. Growth media (1 ml) was then added to each well and the incubationcontinued under the same conditions for 24-48 h. After this period thecell were examined.

In experiments were the condensed DNA contained a reporter gene encodingGreen Fluorescent Protein, several of the cells showed visibleexpression of the reporter protein illustrating successful delivery ofthe DNA into the neuronal cell. Various control experiments wereconducted to confirm the observed transfection in NG108 cells wasreceptor mediated:

Transfection of NG108 cells was found to be dependent on the presence ofmodified clostridial heavy chain fragment within conjugates (notransfection was observed with condensed particles DNA alone)

No transfection was observed in non-neuronal cells (Vero cells) usingthe heavy chain-DNA conjugates.

EXAMPLE 6

Preparation of Conjugates of Modified Clostridial Heavy Chain Fragmentand Microparticles Consisting of Poly(lactide-co-glycolide).

398 mg of poly (lactide co-glycolide) low internal viscosity (3000MW)(Beohringer Mannheim,) was dissolved in 4 ml dichloromethane. Thiswas homogenised at 2000 rpm for 150 seconds with 1 ml of buffer solutioncontaining the therapeutic substance, such as an enzymes and/or drugs.In the case of Mn superoxide dismutase, 10 mg of the enzyme wasdissolved in 10 mM Hepes buffer pH 8.0 containing 100 mM NaCl. Themixture was then added to 50 ml of 8% poly vinyl alcohol and emulsifiedat 2000 rpm for a further 150 seconds. The emulsion was poured into 300ml of ultrapure distilled water at 37° C. and stirred for 30 min at 37°C. The microparticles were collected by centrifugation at 10000×g for 25min at 20° C. and then resuspended in 300 ml water and centrifuged asabove. This washing procedure was the repeated a further 4 times. Afterthe final centrifugation the water supernatant fluid was removed and themicroparticles freeze dried.

2 mg of poly (lactide-co-glycolide) microparticles were re-suspended in1 ml of activation buffer (01 .M MES buffer, pH 6.0 containing 0.5MNaCl). Solid 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) andN-hydroxysulphosuccinimide (sulphoNHS) were added to 2 mM and 5 mMrespectively and the mixture incubated for 15 min at 25° C. Themicroparticles were washed by centrifugation for 1 min at 10000×g andresuspension in 1 ml of activation buffer. The wash step was repeated 4times and then the microparticles resuspended in 1 ml of activationbuffer containing 33 μM of a modified clostridial heavy chain fragmentand incubated for 2 h at 25° C. The reaction was then quenched with 10mM hydroxylamine. After 20 min at 25° C. the microparticles were washedin a suitable buffer by centrifugation as described above.

EXAMPLE 7

Demonstration of the High Affinity Binding to Neuronal Cell TissueDisplayed by Modified Heavy Chain Fragments

Clostridial neurotoxins may be labelled with 125-iodine usingchloramine-T and its binding to various cells assessed by standardmethods such as described in Evans et al. 1986, Eur J. Biochem., 154,409 or Wadsworth et al. 1990, Biochem. J. 268, 123). In theseexperiments the ability of modified clostridial heavy chain constructsto compete with native clostridial neurotoxins for receptors present onneuronal cells or brain synaptosomes was assessed. All bindingexperiments were carried out in binding buffers. For the botulinumneurotoxins this buffer consisted of: 50 mM hepes pH 7.0, 30 mM NaCl,0.25% sucrose, 0.25% bovine serum albumin. For tetanus toxin, thebinding buffer was: 0.05M MES buffer pH 6.0 containing 0.6% bovine serumalbumin. In a typical binding experiment the radiolabelled clostridialneurotoxin was held at a fixed concentration of between 1-10 nM.Reaction mixtures were prepared by mixing the radiolabelled toxin withvarious concentrations of unlabelled neurotoxin or modified clostridialheavy chain construct. The reaction mixture were then added to neuronalcells or rat brain synaptosomes and then incubated at 0-3° C. for 2 hr.After this period the neuronal cells of synaptosomes were washed twicewith binding ice-cold binding buffer and the amount of labelledclostridial neurotoxin bound to cells or synaptosomes was assessed byγ-counting.

In an experiment using a modified clostridial heavy construct whichconsisted of a binding domain derived from tetanus toxin and atranslocation domain from diphtheria toxin, the construct was found tocompete with ¹²⁵I-labelled tetanus neurotoxin for neuronal cellreceptors in a similar manner to unlabelled native tetanus neurotoxin(see FIG. 6). These data showed that the construct had retained bindingproperties of the native neurotoxin.

In a further experiment using Diphtheria H_(N)-BoNT/F H_(c) as themodified clostridial heavy chain, the construct was found to competewith 125I-labelled BoNT/F for receptors on neuronal synaptic membranes(FIG. 7). These data indicate that the modified clostridial heavy chainretains the neuronal receptor-binding properties of BoNT/F.

EXAMPLE 8

Non-Denaturing Gel Electrophoresis to Compare the Sizes of a NativeBotulinum Toxin Heavy Chain (Type A) with that of a Modified ClostridialHeavy Chain (Recombinant Diphtheria H_(N)-BoNT/F H_(C))

Botulinum type A heavy chain was purified as described previously (Shoneet al. 1985 Eur J. Biochemistry 151, 75-82) and recombinant DiphtheriaH_(N)-BoNT/F H_(C) purified as described in Examples 1 and 2. Themodified clostridial heavy chain was purifies as a Maltose BindingProtein fusion with then the fusion protein removed by treatment withFactor Xa. Samples of type A heavy chain (20 μg) and DiphtheriaH_(N)-BoNT/F H_(C) (10 μg) were loaded on a 4-20% Tris-glycinepolyacrylamide gel in Tris-glycine buffer. Samples were electrophoresedto equilibrium (Novex gel system; 43 volts 16 hours) and the gel stainedwith Coomassie blue. The results are shown in FIG. 8. The major band forDiphtheria H_(N)-BoNT/F/H_(C) appears to migrate very close to itspredicted molecular weight of approx 70 kDa. In contrast, the nativetype A heavy chain appears as a diffuse band at approximately 500 kDa,compared to an estimated molecular weight of 100 kDa, which suggestingthe formation of large protein aggregates.

EXAMPLE 9

Recombinant Modified Heavy Chain-Superoxide Dismutase Conjugates.

Recombinant modified heavy chain-superoxide dismutase conjugates wereprepared comprising a combination of the following elements:

-   -   a bacterial superoxide dismutase, from Bacillus        stearothermophilus;    -   a linker region which allows the formation of a disulphide bond        between the superoxide dismutase and the translocation domain        and which also contains a unique protease cleavage site for        cleavage by factor Xa or thrombin to allow the formation of a        dichain molecule;    -   a translocation domain from diphtheria toxin or a endosomolytic        (fusogenic) peptide from influenza virus haemagglutinin); and    -   a neuronal cell-specific binding domain from tetanus or        botulinum neurotoxin type F.

The sequences of these recombinant modified heavy chain-superoxidedismutase conjugates are shown in SEQ ID NO:s 3-7.

To confirm the nature of their structure, the recombinant modifiedclostridial heavy chain-superoxide dismutase conjugates were convertedto the dichain form by treatment with a unique protease corresponding tothe cleavage site sequences within the linker region. Conjugatescontaining the thrombin cleavage site were treated with thrombin (20 μgper mg of conjugate) for 20 h at 37° C.; conjugates containing thefactor Xa cleavage site were treated with factor Xa (20 μg per mg ofconjugate) for 20 min at 22° C.

On SDS-PAGE gels, under non-reducing conditions, the conjugates appearedas a band of molecular mass approx. 120 kDa. In the presence of reducingagent (dithiothreitol) two bands were observed at approx. molecularmasses 70 and 30 kDa corresponding to the modified clostridial heavychain and superoxide dismutase respectively. These data illustrate that,after treatment with the unique protease, the conjugates consist of thelatter two components which are linked by a disulphide bridge.

TABLE 1 Examples of Potential Therapeutic Uses of Modified ClostridialBinding Fragments Therapeutic Substance or Site and Mechanism PotentialEffector of Action Clinical Effects (a) Enzymes:- Superoxide dismutaseReduce oxidative stress Reduction of after stroke/injury of brainneuronal damage or spinal cord after ischemia/ reperfusion Glutaminesynthetase Reduce damage by excess Reduction of glutamate afterstroke/injury neuronal damage of the brain or spinal cord afterischemia/ reperfusion (b) Antibodies:- Anti-tetanus toxin Neutralize theaction of Reverse the effects tetanus toxin at the spinal ofintoxication cord by tetanus toxin Anti SNARE protein Modulateneurotransmitter Hyper secretory (e.g. SNAP-25, VAMPs release disordersSyntaxins) © Viruses/DNA Viral gene Replacement of defective Treatmentof therapy vectors genes within the CNS neurodegenerative (e.g.adenovirus, diseases (Parkinson's' herpes simplex, etc.) Alzheimer's ALSetc.) and other neuronal diseases Non-viral vectors Replacement ofdefective Treatment of for gene therapy genes within the CNSneurodegenerative (e.g. liposomes) diseases and other neuronal diseases(d) Growth factors e.g. BDNF, CTNF, NGF Deliver growth factors toTreatment of the brain and spinal cord neurodegenerative diseases,promotion of neuronal growth after damage (e) Anti-viral agents Deliveranti-viral agents Treatment of latent to the brain or spinal cord viralinfections neurons within neuronal cells, e.g. HIV, herpes simplexinfections (f) Anti-cancer agents Deliver cytotoxic agents Treatment ofneuronal to neoplastic cells of the CNS neoplasia

1. A composition comprising a therapeutic agent linked to a deliverypolypeptide, SEQ ID NO. 12, wherein the delivery polypeptide is fordelivery of a therapeutic agent to a neuronal cell, said deliverypolypeptide comprising: a binding domain that binds to the neuronal celland comprises a hybrid of a botulinum HC domian and a tetanus HC domain,and a translocation domain comprising an HN domain of a diphtheria toxinthat translocates the therapeutic agent into the neuronal cell, whereinthe delivery polypeptide has the binding specificity of tetanus toxinand reduced affinity to neutralizing antibodies to tetanus toxincompared with the affinity to such antibodies of native tetanus toxinheavy chain, and wherein the therapeutic agent is for reduction ofneuronal damage after ischemia/reperfusion or is for promotion ofneuronal growth after damage, and wherein the therapeutic agent is apolypeptide.
 2. The composition of claim 1, wherein the translocationdomain is further a non-aggregating translocation domain as measured bysize in physiological buffers.
 3. The composition of claim 1, whereinthe translocation domain is a non-aggregating translocation domain asmeasured by its size in physiological buffer and wherein the therapeuticagent is for reduction of neuronal damage after ischemia/reperfusion oris for promotion of neuronal growth after damage.
 4. The composition ofclaim 1, wherein the therapeutic agent is chemically bound to saiddelivery polypeptide.
 5. The composition of claim 1 wherein thetherapeutic agent is linked to a translocation domain of said deliverypolypeptide.
 6. The composition of claim 1 wherein the therapeutic agentis an enzyme, growth factor, protein or peptide.
 7. The composition ofclaim 1 wherein the therapeutic agent is produced as a fusion protein byrecombinant technology methods.
 8. A composition comprising atherapeutic agent linked to a delivery polypeptide, SEQ ID NO. 12,wherein the delivery polypeptide is for delivery of a therapeutic agentto a neuronal cell, said delivery polypeptide comprising: a bindingdomain that binds to the neuronal cell and comprises a hybrid of abotulinum HC domian and a tetanus HC domain, and a translocation domaincomprising an HN domain of a diphtheria toxin that translocates thetherapeutic agent into the neuronal cell, wherein the deliverypolypeptide has the binding specificity of tetanus toxin and reducedaffinity to neutralizing antibodies to tetanus toxin compared with theaffinity to such antibodies of native tetanus toxin heavy chain, andwherein the therapeutic agent is for reduction of neuronal damage afterischemia/reperfusion oris for promotion of neuronal growth after damage,and is selected from superoxide dismutase and glutamine synthetase.